A21 -, B30 - modified insulin derivatives having an altered action profile

ABSTRACT

New insulin derivatives, the use thereof, and a pharmaceutical composition containing them 
     Insulin derivatives having an isoelectric point between 5 and 8.5, or physiologically tolerated salts thereof, of the Formula II ##STR1## in which: R 1  at position B1 denotes H or H-Phe; 
     R 2  at position A21 denotes a genetically encodable L-amino acid selected from the group consisting of Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Cys, Tyr, Asp, and Glu; 
     R 30  represents the residue of a neutral genetically encodable L-amino acid selected from the group consisting of Ala, Thr, and Ser; 
     R 31  represents 1, 2, or 3 neutral or basic alpha amino acids, wherein at least one of the alpha amino acids is selected from the group consisting of Arg, Lys, Hyl, Orn, Cit, and His; 
     X represents His at position B10; and 
     the sequences A1 to A20 and B1 to B29 in Formula II correspond to a mammalian insulin; 
     excluding those insulin derivatives in which simultaneously: 
     R 1  at position B1 denotes Phe; and 
     R 3  is one alpha amino acid having a terminal carboxyl group.

This application is a continuation, of application Ser. No. 08/046,481filed Apr. 9, 1993, abandoned, which is a continuation of applicationSer. No. 07/929,510, filed Aug. 19, 1992, abandoned, which is acontinuation of application Ser. No. 07/431,844, filed Nov. 6, 1989, nowabandoned.

BACKGROUND OF THE INVENTION

As is known, insulin and insulin derivatives are required inconsiderable quantities for the treatment of the disease diabetesmellitus, and some of them are also produced on an industrial scale.Despite the considerable number of insulin compositions andmodifications with different action profiles which are already inexistence, there is still a need, because of the variety of organismswith their inter- and intraindividual variations, for other insulinproducts which in turn have other properties and action characteristics.

Insulin derivatives with a delayed action are described, for example, inEP-B 132,769 and EP-B 132,770. These are specifically derivatives with abasic modification in position B31 of the insulin B chain, of thefollowing formula I: ##STR2## in which R¹ denotes H or H-Phe, R³⁰represents the residue of a neutral, genetically encodable L-amino acid,and R³¹ represents a physiologically acceptable organic group which isbasic in nature and has up to 50 carbon atoms, in whose structure 0 to 3α-amino acids are involved and whose terminal carboxyl group which ispresent where appropriate can be free, in the form of an esterfunctionality, an amide functionality, a lactone or reduced to CH₂ OH.

Characteristic of these insulin derivatives is an isoelectric pointbetween 5.8 and 8.5 (measured by isoelectric focusing). The fact thatthe isoelectric point is shifted from the isoelectric point ofunmodified natural insulin or proinsulin (at pH=5.4) into the neutralrange derives from the additional positive charge(s) located on thesurface of the molecule as a result of the basic modification. Thismakes these insulin derivatives with a basic modification less solublein the neutral range than, say, natural insulin or proinsulin, which arenormally dissolved in the neutral range.

The delaying or depot action of the insulin derivatives with a basicmodification, of the formula I, derives from their sparing solubility atthe isoelectric point. According to the two abovementioned publications,the redissolution of the insulin derivatives under physiologicalconditions is achieved by elimination of the additional basic groups,which is brought about, depending on the derivative, by trypsin ortrypsin-like and/or carboxypeptidase B or carboxypeptidase B-like and/oresterase activity. The eliminated groups are in each case either purelyphysiological metabolites or else easily metabolized physiologicallyacceptable substances.

The abovementioned depot principle resulting from basic modification ofthe insulin has also been further utilized by the provision andcorresponding use of other insulin derivatives with basic modifications,mainly within the A and B chains; cf. for example EP-A 0,194,864 andEP-A 0,254,516.

In the insulin derivatives specified in EP-A 0,194,864, a basic aminoacid is incorporated in the B27 position and/or a neutral amino acid islocated at positions A4, A17, B13 and/or B21; in addition, theC-terminal carboxyl group of the B chain is blocked by an amide or esterresidue.

The insulin derivatives specified in EP-A 0,254,516 are very similar tothose specified in the abovementioned EP-A; however, in this case, withthe aim of increasing the stability of the relevant pharmaceuticalcompositions at the weakly acid pH values, the amino acid Ash inposition A21 can also be replaced by other amino acids which are morestable in acid medium, such as, for example, Asp. As is known, Ash(=asparagine) differs from Asp (=aspartic acid) by the blocking of oneof the two carboxyl groups by the amide group: ##STR3##

Rapid-acting insulin derivatives are said to result from yet anothermodification of the insulin molecule in the A and B chain, in particularby replacing the amino acid His, which is responsible for the formationof a complex with zinc--and thus for a certain delaying action, in theB10 position by other appropriate amino acids; cf. EP-A 0,214,826.

All the insulin derivatives specified in the 3 lastmentionedpublications are mainly modified within the A and B chains; they areprepared by genetic engineering routes.

SUMMARY OF THE INVENTION

In the attempt to increase the stability in acid medium of the insulinderivatives with a basic modification on the C-terminal end of the Bchain as specified in the European Patents EP-B 0,132,769 and EP-B0,132,770 mentioned in the introduction, and, where appropriate, also toalter the action profile thereof, it has now been found that this objectis achieved in an advantageous manner by replacing Asn^(A21) by othergenetically encodable amino acids which contain no amide group and,where appropriate, by replacing His^(B10) by other genetically encodableamino acids.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hence the invention relates to insulin derivatives of the formula II##STR4## in which:

R¹ denotes H or H-Phe,

R² denotes a genetically encodable L-amino acid which contains no amidegroup,

R³⁰ represents the residue of a neutral genetically encodable L-aminoacid,

R³¹ represents a physiologically acceptable organic group which is basicin nature and has up to 50 carbon atoms, in whose structure 0 to 3α-amino acids are involved and whose terminal carboxyl group which ispresent where appropriate can be free, in the form of an esterfunctionality, an amide functionality, a lactone or reduced to CH₂ OH,and

X represents a genetically encodable L-amino acid, having an isoelectricpoint between 5 and 8.5, and the physiologically tolerated saltsthereof.

The new insulin derivatives and the physiologically tolerated saltsthereof are stable at the weakly acid pH values of appropriatepharmaceutical compositions even for extended periods andhave--especially when His^(B10) has also been replaced by other aminoacids--an altered (shorter) action profile compared with theknown--unaltered--insulin derivatives with a basic modification of theformula I indicated in the introduction.

R¹ in formula II is preferably H-Phe.

Genetically encodable L-amino acids containing no amide group--for R²--are Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Glu, Cys, Met, Arg, Lys,His, Tyr, Phe, Trp, Pro;

Gly, Ala, Ser, Thr, Asp and Glu are preferred, especially Asp.

Neutral genetically encodable L-amino acids--for R³⁰ --are Gly, Ala,Ser, Thr, Val, Leu, Ile, Ash, Gln, Cys, Met, Tyr, Phe and Pro; Ala, Thrand Set are preferred.

R³¹ is a physiologically acceptable organic group which is basic innature and has up to 50 carbon atoms and in whose structure 0-3 α-aminoacids are involved. When no α-amino acids are involved in the structureof R³¹, examples of suitable basic groups for this residue are thefollowing:

amino-(C₂ -C₆)-alkoxy, (C₁ -C₄)-alkylamino-(C₂ -C₆)-alkoxy, di-(C₁-C₄)-alkylamino-(C₂ -C₆)-alkoxy, tri-(C₁ -C₄)ammonio-(C₂ -C₆)-alkoxy,amino-(C₂ -C₆)-alkylamino, [(C₁ -C₄)-alkyl-amino]- (C₂ -C₆)-alkylamino,di-(C₁ -C₄)-alkylamino-(C₂ -C₆)-alkylamino or [tri-(C₁-C₄)-alkylamino]-(C₂ -C₆)-alkylamino, especially --O--[CH₂ ]--NR₂ and--O--[CH₂ ]--N³⁰ R₃, --NH--[CH₂ ]_(p) --NR₂ or --NH--[CH₂ ]_(p) --⁺ R₃,in which p is 2 to 6, and R is identical or different and representshydrogen or (C₁ -C₄)-alkyl.

When up to 3 α-amino acids are involved in the structure of R³¹ , theseare primarily neutral or basic naturally occurring L-amino acids and/orthe D-amino acids corresponding thereto. Neutral naturally occurringamino acids are, in particular, Gly, Ala, Ser, Thr, Val, Leu, Ile, Ash,Gln, Cys, Met, Tyr, Phe, Pro and Hyp. Basic naturally occurring aminoacids are, in particular, Arg, Lys, Hyl, Orn, Cit and His. If onlyneutral α-amino acids are involved, the terminal carboxyl group thereofcannot be free--in order for R³¹ to be basic in nature; on the contrary,the carboxyl group must in this case be amidated or esterified with abasic group, suitable basic groups for this being, for example, theabovementioned basic groups--in the case where no α-amino acids areinvolved in the structure of R³¹. Of course, these basic ester or amidegroups can also block the carboxyl group of basic α-amino acids. Alsopossible and suitable for blocking the carboxyl group of the basicα-amino acids are--if the blocking is desired--neutral ester or amidegroups such as, for example, (C₁ -C₆)-alkoxy, (C₃ -C₆)-cycloalkyloxy,NH₂, (C₁ -C₆)-alkylamino or di-(C₁ -C₆)-alkylamino.

Of course, the terminal carboxyl group can be in the form of a lactoneonly if the terminal amino acid is a hydroxyamino acid.

Moreover, the terminal carboxyl group can also be reduced to CH₂ OH.

R³¹ is preferably composed of 1, 2 or 3 of the abovementioned basicnaturally occurring amino acids; R³¹ is particularly preferably Arg-OHor Arg-Arg-OH.

Suitable genetically encodable L-amino acids--for X--are the same aminoacids as for R², but the genetically encodable L-amino acids whichcontain an amide group--which are Ash and Gln--are also possible in thiscase; the latter--Asn and Gln--are in fact preferred in this case. IfAsn or Gln is located in position B10, the amide group is at leaststable in weakly acid medium (in contrast to Asn or Gln in positionA21). The sequences (A1-A20) and (B1-B9, B11-B29) are preferably thesequences of human, porcine or bovine insulin, especially the sequencesof human insulin.

Examples of insulin derivatives of the formula II are:

    ______________________________________                                        Asp.sup.A21 -Human insulin-Arg.sup.B31 --OH                                   Glu.sup.A21 -Human insulin-Arg.sup.B31 --OH                                   Gly.sup.A21 -Human insulin-Arg.sup.B31 --OH                                   Ser.sup.A21 -Human insulin-Arg.sup.B31 --OH                                   Thr.sup.A21 -Human insulin-Arg.sup.B31 --OH                                   Ala.sup.A21 -Human insulin-Arg.sup.B31 --OH                                   Asp.sup.A21 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH                     Glu.sup.A21 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH                     Gly.sup.A21 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH                     Ser.sup.A21 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH                     Thr.sup.A21 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH                     Ala.sup.A21 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH                     Asp.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --OH                     Glu.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --OH                     Gly.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --OH                     Ser.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --OH                     Thr.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --OH                     Ala.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --OH                     Asp.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH       Glu.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH       Gly.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH       Ser.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH       Thr.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --Arg.sup.B32 --OH       Ala.sup.A21 --Asn.sup.B10 -Human insulin-Arg.sup.B31 --Arg.sup.B32            ______________________________________                                        --OH                                                                      

The insulin derivatives of the formula II are prepared mainly by agenetic manipulation by means of site-directed mutagenesis usingstandard methods.

For this purpose, a gene structure coding for the desired insulinderivative of the formula II is constructed and its expression isbrought about in a host cell--preferably in a bacterium such as E. colior a yeast, in particular Saccharomyces cerevisiae--and--if the genestructure codes for a fusion protein--the insulinderivative of theformula II is liberated from the fusion protein; analogous methods aredescribed, for example, in EP-A 0,211,299, EP-A 0,227,938, EP-A0,229,998, EP-A 0,286,956 and German Patent Application P 38 21 159.9dated Jun. 23, 1988 (HOE 88/F 158).

After cell disruption, the fusion protein portion is eliminated eitherchemically using cyanogen halide--cf. EP-A 0,180,920 or enzymaticallyusing lysostaphin--cf. DE-A 3,739,347.

The insulin precursor is then subjected to oxidative sulfitolysis by themethod described, for example, by R. C. Marshall and A. S. Inglis in"Practical Protein Chemistry--A Handbook" (edited by A. Darbre) 1986,pages 49-53, and subsequently renatured in the presence of a thiol withthe formation of the correct disulfide bridges, for example by themethod described by G. H. Dixon and A. C. Wardlow in Nature (1960),pages 721-724.

The C peptide is removed by cleavage with trypsin--for example by themethod of Kemmler et al., J. B. C. (1971), pages 6786-6791, and theinsulin derivative of the formula II is purified by known techniquessuch as chromatography--cf., for example, EP-A-0,305,760--andcrystallization.

The insulin derivatives of the formula II with R² =Asp and X=His areexpediently prepared by hydrolysis of the known insulin derivativeswhich have a basic modification and the formula I in aqueous acidicmedium (because only the amide group of the asparagine in position A21must be hydrolyzed in this case), preferably at pH values between about2 and about 4, in particular of about 2.5, and at temperatures of about0° to about 40° C., preferably at room temperature.

The insulin derivatives of the formula II, according to the invention,and/or the physiologically tolerated salts thereof (such as, forexample, the alkali metal or ammonium salts) are mainly used as activesubstances for a pharmaceutical composition for the treatment ofdiabetes mellitus.

The pharmaceutical composition is preferably a solution or suspensionfor injection; it contains at least one insulin derivative of theformula II and/or at least one of the physiologically tolerated saltsthereof in dissolved, amorphous and/or crystalline--preferably indissolved--form.

The composition preferably has a pH between about 2.5 and 8.5, inparticular between about 4.0 and 8.5, and contains a suitable tonicityagent, a suitable preservative and, where appropriate, a suitablebuffer, as well preferably a certain zinc ion concentration, all, ofcourse, in sterile aqueous solution. All the ingredients of thecomposition apart from the active substance form the compositionvehicle.

Examples of suitable tonicity agents are glycerol, glucose, mannitol,NACl, and calcium or magnesium compounds such as CaCl₂, MgCl₂ etc.

The choice of the tonicity agent and/or preservative influences thesolubility of the insulin derivative or the physiologically toleratedsalt thereof at the weakly acid pH values.

Examples of suitable preservatives are phenol, m-cresol, benzyl alcoholand/or p-hydroxybenzoic esters.

Examples of buffer substances which can be used, in particular foradjusting a pH between about 4.0 and 8.5, are sodium acetate, sodiumcitrate, sodium phosphate etc. Otherwise, also suitable for adjustingthe pH are physiologically acceptable dilute acids (typically HCl) oralkalis (typically NaOH).

When the composition contains zinc a content of 1 μg to 2 mg, inparticular from 5 μg to 200 μg, of zinc/ml is preferred.

In order to vary the action profile of the composition according to theinvention it is also possible to admix unmodified insulin, preferablybovine, porcine or human insulin, in particular human insulin.

Preferred concentrations of active substance are those corresponding toabout 1-1500, also preferably about 5-1000, and in particular about40-400, international units/ml.

The invention is now explained in detail by the examples which follow.

A) Preparation By Genetic Manipulation

EXAMPLE 1

Construction of a plasmid for the preparation of Gly (A21)-human insulinArg (B31-OH)

The plasmid pSW3 has been described in German Patent Application P 38 21159.9 (HOE 88/F 158). The plasmid DNA is reacted with the restrictionenzymes PvuII and SalI and subsequently treated with bovine alkalinephosphatase. The two resulting fragments are separated by gelelectrophoresis, and the large fragment is isolated. This fragment islinked in a T4 DNA ligase reaction with the following synthetic DNAsequence: ##STR5##

Competent E. coli W3110 cells are transformed with the ligation mixture.The transformation mixture is plated out on NA plates which contain 20μg of Ap (=Ampicillin)/ml and incubated at 37° C. overnight. Anovernight culture is obtained from single colonies, and plasmid DNA isobtained from this. This DNA is characterized by means of restrictionanalysis and DNA sequence analysis. Correct plasmids which encode themodified A chain are called pIK100. Expression is carried out in analogyto Example 3 of the abovementioned German Patent Application P 38 21159.9. The modified mono-Arg-insulin is likewise prepared in analogy tothe preparation of the unmodified mono-Arg-insulin described in thisGerman Patent Application.

EXAMPLE 2

Construction of a plasmid for the preparation of Ser(A21)-human insulin(Arg B31-OH)

The construction corresponds to the route described in the aboveexample. The synthetic DNA sequence is, however, modified as follows:##STR6##

The plasmid pIK110 which has an additional BspHI recognition sequence isobtained.

EXAMPLE 3

Construction of a plasmid for the preparation of Gly(A21)-Asn(B10)-humaninsulin Arg(B31-OH)

DNA from the plasmid pIK100 is cleaved with the restriction enzymes HpaIand DraIII and treated with bovine alkaline phosphatase. The tworesulting fragments are separated by gel electrophoresis, and the largerof the two fragments is isolated. The fragment is ligated with thesynthetic DNA sequence ##STR7## and competent E. coli W3110 cells aretransformed with the ligation mixture. Further characterization of theresulting plasmid pIK101 is carried out as described in Example 1.

EXAMPLE 4

Construction of a plasmid for the preparation of Ser(A21)-Asn(B10)-humaninsulin

The construction corresponds to the cloning described in Example 3, butstarting from DNA from the plasmid pIK110. The newly constructed plasmidis called pIK111.

EXAMPLE 5

Construction of an expression plasmid for monkey proinsulin

Monkey proinsulin differs from human proinsulin merely by replacement ofa single amino acid in the C peptide (B37-Pro in place of Leu in thisposition of human proinsulin).

The plasmid pSW3 is opened with HpaI and SalI and the remaining plasmidDNA is isolated. The DraIII-SalI monkey proinsulin fragment is isolatedfrom the plasmid pK50 described in EP-A0,229,998. The two fragments arelinked to the synthetic DNA fragment ##STR8## in a T4 DNA ligasereaction. The plasmid pSW2 is obtained, and its DNA is used hereinafteras starting material for the constructions of the expression plasmidsencoding the di-Arg-human insulin derivatives.

EXAMPLE 6

Construction of a plasmid for the preparation of Gly(A21)-human insulinArg(B31)-Arg(B32)-OH

DNA of the plasmid pSW2 is cleaved with PvuII and SalI in accordancewith Example 1 and ligated with the synthetic DNA from Example 1; theresult is the plasmid pSW21.

EXAMPLE 7

Construction of a plasmid for the preparation of Ser(A21)-humaninsulin-Arg(B31)-Arg(B32)-OH

The plasmid pSW22 is constructed starting from pSW2 DNA in analogy toExample 2.

EXAMPLE 8

Construction of a plasmid for the preparation of Gly(A21)-Asn(B10)-humaninsulin-Arg(B31)-Arg(B32)-OH

The plasmid pSW23 is constructed starting from pSW21 DNA in analogy toExample 3.

The following sequence is used as synthetic DNA sequence for this:##STR9##

EXAMPLE 9

Construction of a plasmid for the preparation of Set(A21)-Asn(B10)-humaninsulin-B31(Arg)-B32(Arg)-OH

The plasmid pSW24 is constructed starting from pSW22 DNA in analogy toExample 4 using the synthetic DNA sequence described in Example 8.

B) Preparation of Asp^(A21) -Human Insulin-Arg^(B31) -Arg^(B32) -OH FromHuman Insulin-Arg^(B31) -Arg^(B32) -OH by Hydrolysis

1 g of human insulin-Arg³¹ -Arg³² -OH is suspended in 100 ml of H₂ O.The pH is adjusted to 2.5 by addition of HCl, and the solution is leftat 37° C. After one week about one half of the material has beenconverted into Asp^(A21) -human insulin-Arg^(B31) Arg^(B32) -OH. Theproduct is separated from the starting material in a manner known per seon an anion exchanger, is precipitated from the eluate and iscrystallized in a buffer which contains 10.5 g of citric acid, 1 g ofphenol and 5 ml of a 1% strength zinc chloride solution per liter with aprotein concentration of 5 g/l at pH 6.0. The yield is 390 mg ofAsp^(A21) -human insulin-Arg^(B31) -Arg^(B32).

C) Preparation of an Injection Solution

The insulin derivative from B is dissolved at a concentration of 1.4mg/ml in a sterile vehicle solution of the following composition (perml):

18 mg of glycerol, 10 mg of benzyl alcohol, 80 μg of Zn²⁺, pH 4.0.

D) Action Profile of an Asp^(A21) -Human Insulin-Arg^(B31) -Arg^(B32)-OH Composition in dogs by comparison with human insulin-Arg^(B31)-Arg^(B32) -OH and basal H insulin Hoechst.sup.(R) =an NPH (neutralprotamine Hagedorn) composition containing about 10 μg of Zn²⁺.

    ______________________________________                                                          Blood glucose as a % of the                                                   initial level in hours (h)                                  Product             1 h    2 h   3 h  5 h 7 h                                 ______________________________________                                        According     Asp.sup.A21 -human                                                                          99   62  51   75  98                              to the        insulin                                                         invention     Arg.sup.B31 --Arg.sup.B32 --OH                                                Human insulin 77   52  64   85  98                                            Arg.sup.B31 --Arg.sup.B32 --OH                                  Comparison    Basal H insulin                                                                             71   49  59   83  100                                           Hoechst.sup.(R)                                                 ______________________________________                                    

This example shows that Asp^(A21) -human insulin-Arg^(B31) -Arg^(B32)-OH has the same advantageous basal profile as human insulin-Arg^(B31)-Arg^(B32) -OH. In addition, Asp^(A21) -human-insulin-Arg^(B31)-Arg^(B32) -OH has the advantageous property that the compound is stablefor a long time under the chosen conditions.

I claim:
 1. An insulin derivative having an isoelectric point between 5and 8.5, or a physiologically tolerated salt thereof, of the FormulaIIin which: ##STR10## R¹ at position B1 denotes H or H-Phe; R² atposition A21 denotes a genetically encodable L-amino acid selected fromthe group consisting of Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met,Ser, Thr, Cys, Tyr, Asp, and Glu; R³⁰ represents the residue of aneutral genetically encodable L-amino acid selected from the groupconsisting of Ala, Thr, and Ser; R³¹ represents 1, 2, or 3 neutral orbasic α-amino acids, wherein at least one of the α-amino acids isselected from the group consisting of Arg, Lys, Hyl, Orn, Cit, and His;X represents His at position B10; and the sequences A1 to A20 and B1 toB29 in Formula II correspond to a mammalian insulin; excluding thoseinsulin derivatives in which simultaneously: R¹ at position B1 denotesPhe; and R³¹ is one alpha amino acid having a terminal carboxyl group.2. An insulin derivative or the physiologically tolerated salts thereofas claimed in claim 1, wherein R¹ in formula II represents H-Phe.
 3. Aninsulin derivative or the physiologically tolerated salts thereof asclaimed in claim 1, wherein R² in formula II represents Gly, Ala, Ser,Thr, Asp, or Glu.
 4. An insulin derivative or the physiologicallytolerated salts thereof as claimed in claim 1, wherein R³¹ in formula IIrepresents Arg-Arg-OH.
 5. An insulin derivative or the physiologicallytolerated salts thereof as claimed in claim 1, wherein the sequences (A1to A20) and (B1 to B29) in formula II are the sequences of human,porcine, or bovine insulin.
 6. A pharmaceutical composition thatcontains an effective amount of at least one insulin derivative of theformula II, or at least one of the physiologically tolerated saltsthereof, as claimed in claim 1, in dissolved, amorphous or crystallineform for the treatment of diabetes.
 7. A pharmaceutical composition asclaimed in claim 6, which additionally contains 1 μg to 2 mg of zinc/ml.8. A pharmaceutical composition as claimed in claim 6, whichadditionally contains unmodified insulin.
 9. A method for treating apatient suffering from diabetes mellitus, which comprises administeringto said patient a pharmaceutical composition as claimed in claim
 6. 10.An insulin derivative or the physiologically tolerated salts thereof asclaimed in claim 3, wherein R² in formula II represents Asp.
 11. Aninsulin derivative or the physiologically tolerated salts thereof asclaimed in claim 5, wherein the sequences (A1 to A20) and (B1 to B29) informula II are the sequences of human insulin.
 12. A pharmaceuticalcomposition that contains an effective amount of at least one insulinderivative of the formula II, or at least one of the physiologicallytolerated salts thereof, as claimed in claim 8, in dissolved form forthe treatment of diabetes.
 13. A pharmaceutical composition as claimedin claim 7, which additionally contains 5 μg to 200 μg of zinc/ml.
 14. Apharmaceutical composition as claimed in claim 8, wherein saidunmodified insulin is unmodified human insulin.
 15. An insulinderivative or the physiologically tolerated salts thereof as claimed inclaim 5, wherein R¹ represents H-Phe, R² represents Gly, R³⁰ representsThr, and R³¹ represents Arg-Arg-OH.